The long term goal of this work is to contribute to an understanding of general principles of eukaryotic gene transcription regulatory mechanisms. The gene regulatory system that is being studied here is the galactose-responsive transcription switch, the GAL gene switch, of the small eucaryote, S. cerevisiae. The activities of three proteins, Gal4, Gal80 and Gal3 constitute the primary mechanistic elements of the switch. Gal4, a typical eukaryotic DNA binding transcriptional activator, binds UASgal sites of GAL genes independently of galactose. The Gal4 transcriptional activation domain (Gal4AD) is inhibited in the absence of galactose by binding GalSO. Galactose and ATP activate Gal3 to bind GalSO, relieving GalSO inhibition of Gal4AD and enabling Gal4AD to activate transcription. How the Gal3-Gal80 interaction activates Gal4 is controversial. A widely accepted Non-dissociation model posits that Gal3 binds to a nuclear Gal80-Gal4- UASgal complex to expose the Gal4AD. Our published evidence for GalSO nucleo-cytoplasmic shuttling and Gal80-Gal4 dissociation along with data indicative of Gal80-Gal3 interaction in the cytoplasm challenges the Non-dissociation model. Newer data we have recently published point to the GalSO self- association equilibrium and distinct Gal80 monomer and dimer preferences for binding to Gal3 and Gal4, respectively, as additional features that are not accounted for by a Non-dissociation model. We posit a new model "the GalSO Shuttle" model that specifies that a GalSO monomer binds to activated Gal3 in the cytoplasm and causes;1) a redistribution of GalSO that decreases its concentration in the nucleus, and 2) a reduction in the cellular concentration of the GalSO dimer, the form of GalSO that the model specifies as the form that binds to and inhibits Gal4. The GalSO Shuttle model invokes GalSO nucleo-cytoplasmic rapid shuttling, Gal80-Gal3 binding in the cytoplasm, rapid Gal80 monomer-dimer equilibrium, GalSO monomer and dimer binding preference differences, and dissociation of GalSO from Gal4 as key mechanistic features of the GAL gene switch. The proposed experiments combine genetic, cell biological, and physical methods to test aspects/predictions of the GalSO Shuttle model. All Aims converge on determining how the sub- cellular distributions and dynamics of Gal3 and GalSO integrate with all relevant ligand-protein, protein- protein and protein-DNA interactions to comprise the overall GAL gene switch mechanism. HEALTH RELATEDNESS: this work will provide insight into how individual mechanistic elements involving cellular organization and macromolecular dynamics and interactions are integrated to provide overall principles of transcriptional regulation. Such insight is an important goal for human health care delivery because many disease states, including cancers, are often due to defects in gene regulatory systems. Moreover, what we learn about this gene switch can help design new gene switches for gene expression therapies.